Synthesis of magnetic nanoparticles for nucleic acid purification

ABSTRACT

The present invention relates to monodisperse silanized ferrimagnetic iron oxide particles, a method for producing the same and a method for independent generic binding of nucleic acid molecules to the particles.

CROSS REFERENCE TO RELATED INVENTION

This application claims the benefit of priority under 35 U.S.C. § 119 ofEP 14157699.1, filed Mar. 4, 2014, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to monodisperse silanized ferrimagneticiron oxide particles, a method for producing the same and their use forindependent generic binding nucleic acid molecules.

BACKGROUND OF THE INVENTION

Magnetic nanoparticles are widly used in the field of nucleic acidpurification. All commercially available large scale magneticnanoparticles have superparamagnetic properties. In contrast,ferrimagnetic nanoparticles are not commercially available and onlyknown from academic publications. Such publications includenanoparticles with silica coatings (Chen et al; J. of alloys andcompounds 497 (2010) 221-227; Wang et al; Bioresource Technology 101(2010) 8931-8935; Reza et al; Cent. Eu. J. Chem 5 (2010) 1041-1048). Oneof the major drawbacks of the majority of preparations of ferrimagneticnanoparticles known in the art where low pressure reactors at <100° C.are used is that the synthesis is difficult to upscale and automate(Wang et al; Bioresource Technology 101 (2010) 8931-8935; Reza et al;Cent. Eu. J. Chem 5 (2010) 1041-1048).

The use of glycols as solvent and reducing agent for the synthesis offerrimagnetic nanoparticles has been shown (Wiley et al; Nano Lett. 4(2004) 1733-1739; D. Larcher, R. J. Partrice; J. Solid State Chem. 154(2000) 405-411; Gai et al; J. Phys. D: Appl. Phys. 43 (2010)445-553)—also in combination with the surfactant—free synthesis route(“Green synthesis route”) (Liu et al; Eur. J. Inorg. Chem. 2 (2010)4499-4505). These preparations are focused on the use of iron(III) saltswhich result in poor size distribution and yield when upscaled to morethan 100 mL scale.

Silanization of ferrimagnetic nanoparticles is only known in the artaccording to the Stöber method which relies on harmful alkoxy silanes.Using harmless SiO₂ solution in purely aqueous conditions have only beenshown in the context of superparamagnetic particles (Philipse et al;Langmuir 10 (1994) 92-99).

Extraction of nucleic acids by means of hydrothermally preparedferrimagnetic particle structures are described (Gai et al; J. Phys. D:Appl. Phys. 43 (2010) 445-553). These publications used ferrimagneticparticles which were produced by silane chemistry or without siliconecontaining coating, wherein the production was complex or resulted in apoor eluation.

The object of the present invention is the provision of monodispersesilanized ferrimagnetic iron oxide particles for nucleic acid bindingwhich do not show the above mentioned drawbacks and a method forproducing the same.

SUMMARY OF THE INVENTION

It was found that silanized ferrimagnetic iron oxide particles forindependent generic nucleic acid binding can be produced with a highdegree of uniformity (very defined reproducible diameters of theparticles), a high yield and homogeniously high magnetizationsaturations. Furthermore it was found that silanizing can be performedresulting in a very dense layer of silicate around the magnetite core ofthe particles, such that the quality of silanization is compareable tothe quality reached by known processes, however, with less harmfulchemicals and more cost effective educts as compared to the knownprocesses.

The present invention thus relates to a method for producing a pluralityof silanized ferrimagnetic iron oxide particles for independent genericnucleic acid binding, wherein the method comprises the steps of a)adding an iron(II) salt to a liquid glycol to obtain a solution, b)raising the pH of the solution to a value of at least 9 such that aprecipitate is obtained, wherein during steps a) and b) a firsttemperature is applied to the solution and wherein during steps a) andb) the solution is gassed with nitrogen, c) mixing the solutioncomprising the precipitate at a second temperature such thatferrimagnetic iron oxide particles are obtained, and d) contacting theferrimagnetic iron oxide particles with a silicate solution such thatsilanized ferrimagnetic iron oxide particles are obtained. In oneembodiment the step of contacting the ferrimagnetic iron oxide particleswith a silicate solution comprises the steps of d1) sonificating thesilicate solution comprising the ferrimagnetic iron oxide particles, d2)lowering the pH of the silicate solution to a value of 6 or below suchthat silanized ferrimagnetic iron oxide particles are obtained, d3)washing of the silanized ferrimagnetic particles with water, and d4)washing of the silanized ferrimagnetic particles with isopropanol suchthat crosslinking occurs within the silicate layer.

The present invention further relates to monodisperse silanizedferrimagnetic iron oxide particles for independent generic nucleic acidbinding comprising a core, wherein the core comprises an inner layercomprising Fe₃O₄ and an outer layer comprising Fe₂O₃, a coating, whereinthe coating comprises silica and silicates from sodium silicateprecipitation, wherein the particles display a sedimentation speed inpure water of less than 60 μm/s, and no significant iron bleedingparticles in 1M HCl for at least 60 min.

The present invention further relates to a method for independentgeneric binding of nucleic acid molecules to a composition comprising,contacting a sample containing nucleic acid molecule to the composition,wherein the composition comprises monidisperse silanized ferrimagneticiron oxice particles, wherein said particles comprise a core, whereinthe core comprises an inner layer comprising Fe₃O₄ and an outer layercomprising Fe₂O₃, a coating, wherein the coating comprises silica andsilicates from sodium silicate precipitation, wherein the particlesdisplay a sedimentation speed in pure water of less than 60 μm/s, and nosignificant iron bleeding in 1M HCl for at least 60 minutes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Shows the regulation temperature and the resulting temperature ofthe reactor.

FIG. 2 Shows the labeled parts of the disassembled stirrer.

FIG. 3 Shows the manometer, temperature sensor, N₂-pipe, needle valveand burst protection and corresponding parts.

FIG. 4 Shows the core of the top panel and corresponding parts.

FIG. 5 Schematic drawing of the special design glas reactor used forsilanizing the ferrimagnetic iron oxide particles. (Ar=Argon, M=Motor,US=Ultrasonic probe).

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are set forth to illustrate and define themeaning and scope of various terms used to describe the inventionherein.

The terms “a”, “an” and “the” generally include plural referents, unlessthe context clearly indicates otherwise.

The term “acid” is used herein as known to the expert skilled in the artand refers to a substance capable of donating a proton in polar ornon-polar solvents. The acid of choice for a particular reaction dependson the starting materials, the solvent and the temperature used for aspecific reaction. Examples of acids include phosphoric acid, sulphuricacid and hydrochloric acid.

The term “alkyl” denotes a monovalent linear or branched saturatedhydrocarbon group of 1 to 12 carbon atoms. In particular embodiments,alkyl has 1 to 7 carbon atoms, and in more particular embodiments 1 to 4carbon atoms. Examples of alkyl include methyl, ethyl, propyl,isopropyl, n-butyl, iso-butyl, sec-butyl, or tert-butyl.

In the context of the pH the below terms are defined as follows:

-   -   “At least”: A “pH of at least 7” refers to a pH value of 7 or        higher, e.g. a pH value of 7, 8, 9, 10, 11, 12, 13 or 14.    -   “Raise”: “Raising the pH” refers to changing the pH from a lower        value to a higher value, e.g. changing the pH from 7 to 8.    -   “Or below”: A “pH of 7 or below” refers to a pH value of 7 or        lower, e.g. a pH value of 1, 2, 3, 4, 5, 6 or 7.    -   “Lower”: “Lowering the pH” refers to changing the pH from a        higher value to a lower value, e.g. changing the pH from 8 to 7.

The term “base” is used herein as known to the expert skilled in the artand refers to a substance capable of accepting a proton in polar ornon-polar solvents. The base of choice for a particular reaction dependson the starting materials, the solvent and the temperature used for aspecific reaction. Examples of bases include carbonate salts such aspotassium carbonate, potassium bicarbonate, sodium carbonate, sodiumbicarbonate, and cesium carbonate; halides such as cesium fluoride;phosphates such as potassium phosphate, potassium dihydrogen phosphate,and potassium hydrogen phosphate; hydroxides such as lithium hydroxide,sodium hydroxide, and potassium hydroxide; disilylamides such as lithiumhexamethyldisilazide, potassium hexamethyldisilazide, and sodiumhexamethyldisilazide; trialkylamines such as triethylamine,diisopropylamine, and diisopropylethylamine; heterocyclic amines such asimidazole, pyridine, pyridazine, pyrimidine, and pyrazine; bicyclicamines such as DBN and DBU; and hydrides such as lithium hydride, sodiumhydride, and potassium hydride. Examples of bases include alkali metalhydroxides as defined herein; alkali metal hydrides such as lithium,sodium, or potassium hydride; and nitrogen-containing bases such aslithium diisopropyl amide (LDA), lithium bis(trimethylsilyl)amide,sodium bis(trimethylsilyl)amide, and potassium bis(trimethylsilyl)amide;and the like. It will be apparent to a skilled practioner thatindividual base and solvent combinations can be preferred for specificreaction conditions depending on such factors as the solubility ofreagents, reactivity of reagents with Isomidazolam or the solvent, andpreferred temperature ranges.

The term “crosslinking” as used herein refers to a chemical process,wherein two more molecules interact to form n-mers, wherein n>1. Suchinteraction can be a covalent bond, hydrophilic or hydrophobic, ionic orelectrostatic interaction.

The term “ferrimagnetic” as used herein refers to a material consistingof populations of atoms with opposing but unequally distributed magneticmoments.

Thus resulting in a magnetic saturation and remanence once an externalmagnetic field was applied.

The term “independent generic nucleic acid binding” as used hereinrefers to binding of single stranded and/or double stranded nucleic acidmolecules such as DNA and/or RNA. Binding of such molecules occurindependently of properties of the nucleic acid molecules, suchsequence, presence or absence of modifications and concentration.

The term “iron bleeding” as used herein refers to the solvation of ironions into the surrounding solvation medium. The term “significant ironbleeding” as used herein refers to a iron ion concentration in thesurrounding solvent detectable by light spectroscopic methods(UV/Vis-Spectroscopy). Spectroscopy was performed from 200 nm to 800 nmin a quarz glas cuvette with 1 cm transmission distance in a totalvolume of 3 mL 5% w/w particle after adding 5M HCl solution and shakingfor 10 seconds followed by magnetic separation of the beads by using aNeodymium-Iron-Borum magnet (1 cm×1 cm×1 cm). After the blanking withpure water no peak larger than the background noise should be seen oridentified by the analysis software over the whole spectrum.

The term “monodisperse” as used herein refers to particles ofessentially the same size. The size of the monodisperse silanizedferrimagnetic iron oxide particles of one certain batch is essentiallythe same for all particles within that batch. Essentially the same sizeof the particles has to be interpreted in the context of thisdescription such that the difference in size, i.e. difference indiameter, of the particles is in average smaller than 5% (coefficient ofvariation).

The terms “nucleic acid”, “nucleic acid molecule” or “polynucleotide”can be used interchangeably and refer to a polymer that can becorresponded to a ribose nucleic acid (RNA) or deoxyribose nucleic acid(DNA) polymer, or an analog thereof. This includes polymers ofnucleotides such as RNA and DNA, as well as synthetic forms, modified(e.g., chemically or biochemically modified) forms thereof, and mixedpolymers (e.g., including both RNA and DNA subunits). Exemplarymodifications include methylation, substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, carbamates, and the like), pendentmoieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen,and the like), chelators, alkylators, and modified linkages (e.g., alphaanomeric nucleic acids and the like). Also included are syntheticmolecules that mimic polynucleotides in their ability to bind to adesignated sequence via hydrogen bonding and other chemicalinteractions. Typically, the nucleotide monomers are linked viaphosphodiester bonds, although synthetic forms of nucleic acids cancomprise other linkages (e.g., peptide nucleic acids as described inNielsen et al. (Science 254:1497-1500, 1991). A nucleic acid can be orcan include, e.g., a chromosome or chromosomal segment, a vector (e.g.,an expression vector), an expression cassette, a naked DNA or RNApolymer, the product of a polymerase chain reaction (PCR), anoligonucleotide, a probe, and a primer. A nucleic acid can be, e.g.,single-stranded, double-stranded, or triple-stranded and is not limitedto any particular length. Unless otherwise indicated, a particularnucleic acid sequence comprises or encodes complementary sequences, inaddition to any sequence explicitly indicated.

The term “plurality” as used herein refers to two or more items orcomponents. Accordingly, the term “plurality of particles” refers to twoor more particles, such as silanized ferromagnetic iron oxide particles.

As used herein, the term “precipitate” refers to the formation of solid,such as a particle, in a solution during a chemical reaction.

The term “sedimentation speed” as used herein refers to the speed(length/time) with which a particle, such as a silanized ferromagneticiron oxide particle, sediments at a defined gravitational force within aliquid, such as pure water. If no value for a defined gravitationalforce is given, the gravitational force to be assumed for the givenreaction is gravitation of earth, i.e. 1.0 g.

The term “silanized” as used herein refers to the formation of a toplayer containing silicone which is crosslinked by oxygen.

In one aspect, the description refers to a method for producing aplurality of silanized ferrimagnetic iron oxide particles forindependent generic nucleic acid binding, wherein the method comprisesthe steps of a) adding an iron(II) salt to a liquid glycol to obtain asolution, b) raising the pH of the solution to a value of at least 9such that a precipitate is obtained, wherein during steps a) and b) afirst temperature is applied to the solution and wherein during steps a)and b) the solution is gassed with nitrogen, c) mixing the solutioncomprising the precipitate at a second temperature such thatferrimagnetic iron oxide particles are obtained, and d) contacting theferrimagnetic iron oxide particles with a silicate solution such thatsilanized ferrimagnetic iron oxide particles are obtained.

The addition of the iron(II) salt to the liquid glycol to obtain asolution as performed in step a) above has the advantage over themethods known in the art that it results in a better size control.Furthermore, gassing the solution with nitrogen leads to a higher yield.

In one embodiment, the step of contacting the ferrimagnetic iron oxideparticles with a silicate solution comprises the steps of d1)sonificating the silicate solution comprising the ferrimagnetic ironoxide particles, d2) lowering the pH of the silicate solution to a valueof 6 or below such that silanized ferrimagnetic iron oxide particles areobtained, d3) washing of the silanized ferrimagnetic particles withwater, and d4) washing of the silanized ferrimagnetic particles withisopropanol such that crosslinking occurs within the silicate layer.

The crosslinking performed in step d4) results in a very dense layer ofsilicate around the magnetite core and in a silanization compareable tothe Stöber method. However, the crosslinking according to the presentdescription is performed with harmless chemicals and cost effectiveeducts.

In one embodiment, the iron(II) salt is soluble in the liquid glycol. Ina specific embodiment, the iron(II) salt soluble in the liquid glycol isselected from the group consisting of FeCl₂, FeSO₄, FeAc₂ and thehydrated forms thereof. The term hydrated forms as used herein has to beunderstood as a compound which is formed by the addition of water.Possible hydrated forms according to the present description are FeCl₂.nH₂O, FeSO₄.n H₂O, FeAc₂.n H₂O, wherein n≥1. Specific hydrated formsaccording to the present description are FeCl₂.4H₂O, FeSO₄.4H₂O,FeAc₂.4H₂O. In a specific embodiment, the iron(II) salt soluble in theliquid glycol is FeCl₂ and hydrated forms thereof.

In one embodiment, the concentration of FeCl₂.4H₂O in the liquid glycolis from 50 mmol to 70 mmol. In a specific embodiment, the concentrationof FeCl₂.4H₂O in the liquid glycol is from 55 mmol to 65 mmol. In a morespecific embodiment, the concentration of FeCl₂.4H₂O in the liquidglycol is from 58 mmol to 62 mmol. In a specific embodiment, theconcentration of FeCl₂.4H₂O in the liquid glycol is 61.9 mmol.

In a specific embodiment, the liquid glycol is an alkyl glycol andpolymerized forms thereof. In a specific embodiment, the alkyl glycol isselected from the group consisting of ethylene glycol, diethyleneglycol, triethylene glycol, tetraethylene glycol, propylene glycol,dipropylene glycol, tripropylene glycol, tetraproylene glycol, butyleneglycol, dibutylene glycol, tributylene glycol, tetrabutylene glycol. Ina more specific embodiment, the alkyl glycol is triethylene glycol.

In one embodiment, the pH of the solution in step b) is raised to avalue of at least 9. In a specific embodiment, the pH of the solution instep b) is raised to a value of at least 10. In a more specificembodiment, the pH of the solution in step b) is raised to a value of10.5.

In one embodiment, the pH of the solution in step b) is raised using abase. In a specific embodiment, the pH of the solution in step b) israised using an alkaline metal hydroxide. In a more specific embodiment,the pH of the solution in step b) is raised using an alkaline metalhydroxide selected from the group consisting of lithium hydroxide,sodium hydroxide and potassium hydroxide.

In one embodiment, the first temperature has a value from 20 to 120° C.In a specific embodiment, the first temperature has a value from 40 to120° C., from 60 to 120° C., from 80 to 120° C., or from 90 to 110° C.In a more specific embodiment, the first temperature has a value from 95to 105° C. In an even more specific embodiment, the first temperaturehas a value of 100° C. The application of the first temperature resultsin the evaporation of excess water from the reaction and thus in highermagnetization rates due to less uncontrolled further oxidation of themagnetite produced during the reaction.

In one embodiment, the first temperature is maintained for 5 to 60 min.In specific embodiment, the first temperature is maintained for 10 to 50min. In a more specific embodiment, the first temperature is maintainedfor 20 to 50 min, for 30 to 50 min or for 35 to 45 min. In an even morespecific embodiment, the first temperature is maintained for 40 min.

In one embodiment the gassing with nitrogen is performed at a pressureof between 0.1 and 1.0 bar at the outlet valve. In a specificembodiment, the gassing with nitrogen is performed at a pressure ofbetween 0.2 and 0.8 bar at the outlet valve. In a more specificembodiment, the gassing with nitrogen is performed at a pressure ofbetween 0.3 and 0.6 bar at the outlet valve. In an even more specificembodiment, the gassing with nitrogen is performed at a pressure ofbetween 0.35 and 0.45 bar at the outlet valve. In a specific embodiment,the gassing with nitrogen is performed at a pressure of 0.4 bar at theoutlet valve. Gassing with nitrogen at the above mentioned pressurevalues reduces the oxygen present in the solution and thus reducesundesirable oxidation during precipitation of the particles. Thus,higher yields and homogeniously high magnetization saturations areachieved.

In one embodiment, the second temperature has a value from 150 to 300°C. In a specific embodiment, the second temperature has a value from 200to 300° C., from 210 to 290° C., from 220 to 280° C., from 230 to 270°C. or from 240 to 260° C. In a more specific embodiment, the secondtemperature has a value of 250° C. The second temperature results inparticles with defined reproducible diameters even at scales larger than500 mL.

In one embodiment, the second temperature is maintained for 20 min to 48h. In a specific embodiment, the second temperature is maintained for 20min to 40 h, for 20 min to 30 h, for 20 min to 20 h, for 20 min to 10 h,for 20 min to 5 h, for 20 min to 2 h, for 30 min to 90 min, for 40 minto 90 min, for 50 min to 90 min, for 60 min to 90 min or for 70 min to90 min. In a more specific embodiment, the second temperature ismaintained for 80 min.

In a specific embodiment, the second temperature is regulated accordingto the following protocol. The second temperature starts at a value of80° C. and is increased to a value of 250° C. within a time period of 20min. The value of 250° C. is maintained for a time period of 80 min.Subsequently, the second temperature is decreased to a value of 30° C.within a time period of 35 min. The second temperature refers to thetemperature within the reactor. The temperature gradient within thereactor and the actual regulation temperature is depicted in FIG. 1. Thegradient as shown in the figure has to be accurately executes such thatthe controlled environment ensures the control of the reaction forforming particles with a homogeneous size distribution and highmagnetization saturations.

In one embodiment, the silicate is selected from the group consisting ofpotassium silicate and sodium silicate. In a specific embodiment, thesilicate is sodium silicate.

In one embodiment, the silicate is present in the solution in aconcentration from 1 to 30% w/v. In a specific embodiment, the silicateis present in the solution in a concentration from 1 to 20% w/v, from 1to 15% w/v, from 1 to 15% w/v, from 1 to 10% w/v, from 3 to 7% w/v orfrom 4 to 6% w/v. In a specific embodiment, the silicate is present inthe solution in a concentration of 5.3% w/v.

In one embodiment, sonificating is performed with an amplitude of 60 to100%. In a specific embodiment, sonificating is performed with anamplitude of 70 to 90%. In a more specific embodiment, sonificating isperformed with an amplitude of 75 to 85%. In an even more specificembodiment, sonificating is performed with an amplitude of 80%. In oneembodiment, sonificating is performed with a cycle of 10 to 30%. In aspecific embodiment, sonificating is performed with a cycle of 15 to25%. In a more specific embodiment, sonificating is performed with acycle of 18 to 22%. In an even more specific embodiment, sonificating isperformed with a cycle of 20%. In a specific embodiment, sonificating isperformed with 80% of amplitude and 20% of cycle.

In one embodiment, sonificating is performed for a duration of 1 to 10seconds. In a specific embodiment, sonificating is performed for aduration of 3 to 8 seconds. In a more specific embodiment, sonificatingis performed for a duration of 4 to 6 seconds. In an even more specificembodiment, sonificating is performed for a duration of 5 seconds. Inone embodiment, the sonificating pulses are interrupted by a pause of 30to 70 seconds. In a specific embodiment, the sonificating pulses areinterrupted by a pause of 40 to 60 seconds. In a more specificembodiment, the sonificating pulses are interrupted by a pause of 45 to55 seconds. In an even more specific embodiment, the sonificating pulsesare interrupted by a pause of 50 seconds. In a specific embodiment,sonificating is performed for a duration of 5 seconds followed by apause of 50 seconds.

In one embodiment, the pH of the solution in step d2) is lowered to avalue of 7 or below. In a specific embodiment, the pH of the solution instep d2) is lowered to a value of 6 or below. In a more specificembodiment, the pH of the solution in step d2) is lowered to a value of5.

In one embodiment, the pH of the solution in step d2) is lowered usingan acid selected from the group consisting of phosphoric acid, sulphuricacid and hydrochloric acid. In a specific embodiment, the pH of thesolution in step d2) is lowered using any non-oxidizing andwater-soluble acid. In a specific embodiment, the pH of the solution instep d2) is lowered using a non-oxidizing and water-soluble acidselected from the group consisting of hydrochloric acid, boric acid orprussic acid. In a specific embodiment, the pH of the solution in stepd2) is lowered using hydrochloric acid. In another specific embodiment,the pH of the solution in step d2) is lowered using 1M hydrochloricacid.

In another embodiment, the method for producing a plurality of silanizedferrimagnetic iron oxide particles for independent generic nucleic acidbinding comprises the steps of a) adding FeCl₂ or the hydrated formsthereof to triethylene glycol to obtain a solution, b) raising the pH ofthe solution to a value of 10.5 with sodium hydroxide such that aprecipitate is obtained, wherein during steps a) and b) a firsttemperature is applied to the solution, wherein the first temperaturehas a value of 100° C. and is maintained for 40 min, and wherein duringsteps a) and b) the solution is gassed with nitrogen at a pressure of0.4 bar, c) mixing the solution comprising the precipitate at a secondtemperature, wherein the second temperature has a value of 250° C. andis maintained for 80 min, such that ferrimagnetic iron oxide particlesare obtained, and d) contacting the ferrimagnetic iron oxide particleswith a solution of 5.3% w/v sodium silicate such that silanizedferrimagnetic iron oxide particles are obtained, wherein the step ofcontacting the ferrimagnetic iron oxide particles with the silicatesolution comprises the steps of d1) sonificating the silicate solutioncomprising the ferrimagnetic iron oxide particles, d2) lowering the pHof the silicate solution to a value of 5 such that silanizedferrimagnetic iron oxide particles are obtained, d3) washing of thesilanized ferrimagnetic particles with water, and d4) washing of thesilanized ferrimagnetic particles with isopropanol such thatcrosslinking occurs within the silicate layer.

In some embodiments of the method described herein, step d) is repeatedone or more times. Between the respective repetitions, the ferrimagneticiron oxide particles are washed with a wash solution. Hence, in someembodiments, the method described herein comprises after step d) theadditional step e): washing the silanized ferrimagnetic iron oxideparticles one or more times with a wash solution. In some embodiments,the wash buffer in step e) is water, in some embodiments distilled ordeionized water. In further embodiments, the wash buffer is isopropanolor an equivalent alcohol. The sequence of steps d) and e) is in someembodiments repeated one or more times. Where step d) comprises stepsd1) to d4), the above-mentioned embodiments apply to the respectivesequence of substeps from d1) to d4).

The repetition of step d) or the sequence of steps d) and e),respectively, result in particles with multiple layers of silica andsilicates from sodium silicate precipitation. As shown in Example 4,such multiple-layered silanized ferrimagnetic iron oxide particlesdisplay enhanced nucleic acid binding properties.

In another aspect, the present invention relates to monodispersesilanized ferrimagnetic iron oxide particles for independent genericnucleic acid binding having the following characteristics: A core,wherein the core comprises an inner layer comprising Fe₃O₄ and an outerlayer comprising Fe₂O₃, a coating, wherein the coating comprises silicaand silicates from sodium silicate precipitation, a sedimentation speedin pure water of less than 60 μm/s, and no significant iron bleedingoccurs on the particles in 1M HCl for at least 60 min.

In one embodiment, the difference it size of monodisperse silanizedferrimagnetic iron oxide particles is in average smaller than 5%. In aspecific embodiment, the size of the particles is between 20 nm to 600nm. In another embodiment, the size of the particles is 100 nm. In stillanother embodiment, the diameter of the particles is 100 nm. The size ofthe particles of one certain batch can be varied by adjusting theconcentration of iron(II) salt in the liquid glycol. This however has tobe understood that the size of the silanized ferrimagnetic iron oxideparticles of one certain batch is essentially the same for all particleswithin that batch. Essentially the same size of the particles has to beinterpreted in the context of this description such that the differencein size of the particles is in average smaller than 5%.

The monodisperse silanized ferrimagnetic iron oxide particles accordingto the description appear black in suspension. In one embodiment, thesedimentation speed of the monodisperse silanized ferrimagnetic ironoxide particles in pure water is less than 100 μm/s, less than 90 μm/s,less than 80 μm/s, less than 70 μm/s, less than 60 μm/s or less or equalthan 50 μm/s. In a specific embodiment, the sedimentation speed of themonodisperse silanized ferrimagnetic iron oxide particles in pure wateris 50 μm/s.

In one embodiment, the yield of the synthesis of the monodispersesilanized ferrimagnetic iron oxide particles is at least 20%, at least30%, at least 40%, at least 50%, at least 60% or at least 70%. In aspecific embodiment, the yield of the synthesis of the monodispersesilanized ferrimagnetic iron oxide particles is at least 75%.

As an alternative or an addition, for monodisperse silanizedferrimagnetic iron oxide particles that have multiple layers of coatingas described in the context of the production method, the relativeamount of coating on a specific particle or population of particles maybe varied. For instance, the mass of coating may be, for instance, 20%,40%, 60%, 80%, 100%, 120%. 140%, 160%, 180%, 200%, or other percentagesrelative to the mass of the respective iron oxide core. Such particlesare used in Example 4.

In another aspect, the present invention relates to the use of themonodisperse silanized ferrimagnetic iron oxide particles as describedin the previous paragraph for independent generic binding nucleic acidmolecules. In one embodiment, the nucleic acid molecules are selectedfrom the group consisting of RNA molecules and DNA molecules. In aspecific embodiment, the nucleic acid molecules are DNA molecules. In afurther embodiment, the nucleic acid molecules are RNA molecules.

As described supra, the binding of nucleic acids to magnetic particlesis often part of a nucleic acid isolation or extraction procedure fromessentially any source, such as cultured microorganisms, unculturedmicroorganisms, complex biological mixtures, tissues, sera, ancient orpreserved tissues, environmental isolates or the like or from any“sample” that contains nucleic acid. Typically, one of the first stepsof purification of a biological target material comprises releasing thecontents of cells or viral particles by using enzymes and/or chemicalreagents. This process is commonly referred to as “lysis”. The nucleicacid to be isolated is ideally essentially unaffected by the lysis step.As known in the art, lysis procedures can involve chaotropic agents,ionic and/or non-ionic detergents such as SDS or sarcosyl, mechanicdisruption by shearing forces or the like, French Press, ultrasound,liquid nitrogen, enzymes such as lysozyme or proteases, freeze-drying,heat or osmotic shock, cell membrane disruption under alkalineconditions, or other measures known by the person skilled in the art.

The released nucleic acid may then be bound to suitable bindingparticles such as the silanized ferrimagnetic iron oxide particlesdescribed herein. This binding step may involve the presence ofchaotropic agents.

“Chaotropic agents” are substances that generally disturb the orderedstructure of water molecules in solution and non-covalent binding forcesin and between molecules. They can make several contributions to theisolation procedure. Chaotropic agents also contribute to the disruptionof biological membranes, such as plasma membranes or the membranes ofcell organelles, if present. Useful chaotropic agents in the context ofthe present invention include, but are not limited to, guanidinium saltssuch as guanidinium thiocyanate, guanidinium hydrochloride, guanidiniumchloride or guanidinium isothiocyanate, urea, perchlorates such aspotassium perchlorate, other thiocyanates or potassium iodide or sodiumiodide. They can be applied as RNase inhibitors by disturbing thenuclease's tertiary structure. Usually, no further RNase inhibitor needto be applied to the lysis buffer when the biological target material isa nucleic acid. Also, chaotropic agents can play a significant role inthe adhesive binding of nucleic acids to surfaces like glass. For lysisand/or binding purposes, chaotropic agents can be applied at aconcentration of about 2 to about 8 M, and in some embodiments at aconcentration of about 4 to about 6 M.

Further embodiments are included by the following items:

-   1. Method for producing a plurality of silanized ferrimagnetic iron    oxide particles for independent generic nucleic acid binding,    wherein the method comprises the steps of:-   a. Adding an iron(II) salt to a liquid glycol to obtain a solution,-   b. Raising the pH of the solution to a value of at least 9 such that    a precipitate is obtained,    -   wherein during steps a) and b) a first temperature is applied to        the solution and wherein during steps a) and b) the solution is        gassed with nitrogen,-   c. Mixing the solution comprising the precipitate at a second    temperature such that ferrimagnetic iron oxide particles are    obtained, and-   d. Contacting the ferrimagnetic iron oxide particles with a silicate    solution such that silanized ferrimagnetic iron oxide particles are    obtained.-   2. The method of item 1, wherein contacting the ferrimagnetic iron    oxide particles with a silicate solution comprises the steps of:-   d1. Sonificating the silicate solution comprising the ferrimagnetic    iron oxide particles,-   d2. Lowering the pH of the silicate solution to a value of 6 or    below such that silanized ferrimagnetic iron oxide particles are    obtained,-   d3. Washing of the silanized ferrimagnetic particles with water, and-   d4. Washing of the silanized ferrimagnetic particles with    isopropanol such that crosslinking occurs within the silicate layer.-   3. Method of items 1-2, wherein the iron(II) salt is soluble in the    liquid glycol.-   4. Method of item 3, wherein the iron(II) salt soluble in the liquid    glycol is selected from the group consisting of FeCl₂, FeSO₄, FeAc₂    and the hydrated forms thereof-   5. Method of items 1 to 4, wherein the liquid glycol is an alkyl    glycol and polymerized forms thereof-   6. Method of item 5, wherein the alkyl glycol is selected from the    group consisting of ethylene glycol, diethylene glycol, triethylene    glycol, tetraethylene glycol, propylene glycol, dipropylene glycol,    tripropylene glycol, tetraproylene glycol, butylene glycol,    dibutylene glycol, tributylene glycol, tetrabutylene glycol.-   7. Method of item 5, wherein the alkyl glycol is triethylene glycol.-   8. Method of items 1-7, wherein the pH of the solution in step b) is    raised to a value of 10.5.-   9. Method of items 1-8, wherein the pH of the solution in step b) is    raised using sodium hydroxide.-   10. Method of items 1-9, wherein the first temperature has a value    from 20 to 120° C.-   11. Method of item 10, wherein the first temperature has a value of    100° C.-   12. Method of item 11, wherein the first temperature is maintained    for 5 to 60 min.-   13. Method of items 1-12, wherein the second temperature has a value    from 150 to 300° C.-   14. Method of item 13, wherein the second temperature has a value of    250° C.-   15. Method of item 14, wherein the second temperature is maintained    for 20 min to 48 h.-   16. Method of items 1-15, wherein the silicate is selected from the    group consisting of potassium silicate and sodium silicate.-   17. Method of items 1-16, wherein the silicate is present in the    solution in a concentration from 1 to 30% w/v.-   18. Method of item 17, wherein the silicate is present in the    solution in a concentration of 5.3% w/v.-   19. Method of items 2-18, wherein the pH of the solution in step d2)    is lowered to a value of 5.-   20. Method of item 19, wherein the pH of the solution in step d2) is    lowered using an acid selected from the group consisting of    phosphoric acid, sulphuric acid and hydrochloric acid.-   21. Method of item 20, wherein the pH of the solution in step d2) is    lowered using hydrochloric acid.-   22. Method of items 1-21, wherein step d) is repeated one or more    times.-   23. Monodisperse silanized ferrimagnetic iron oxide particles for    independent generic nucleic acid binding having the following    characteristics:-   a. A core, wherein the core comprises an inner layer comprising    Fe₃O₄ and an outer layer comprising Fe₂O₃,-   b. A coating, wherein the coating comprises silica and silicates    from sodium silicate precipitation,-   c. A sedimentation speed in pure water of less than 60 μm/s, and-   d. No significant iron bleeding occurs on the particles in 1M HCl    for at least 60 min.-   24. The monodisperse silanized ferrimagnetic iron oxide particles    according to item 23, wherein the difference in size of the    monodisperse silanized ferrimagnetic iron oxide particles is in    average smaller than 5%.-   25. The monodisperse silanized ferrimagnetic iron oxide particles    according to item 24, wherein the size of the particles n is between    20 nm and 600 nm.-   26. Method for independent generic binding of nucleic acid molecules    by use of the monodisperse silanized ferrimagnetic iron oxide    particles of items 23-25.-   27. Method of item 26, wherein the nucleic acid molecules are DNA    molecules or RNA molecules.

EXAMPLES

The following examples 1 to 4 are provided to aid the understanding ofthe present invention, the true scope of which is set forth in theappended claims. It is understood that modifications can be made by aperson of ordinary skill in the art to the procedures set forth below.

Example 1 Cleaning of the Reactor

The reactor used in the present description is a Büchi Midiclave Reactorwith propeller stirrer and stream breaker from Hasteloy Steel.

Immediately after the removal of the ironoxide nanoparticle suspension,the flow break, agitator shaft and temperature sensor were washed 3times with water.

The reactor was filled with 900 mL of a 0.02 M EDTA and 0.0067 M FeCl₂solution and was heated to 120° C. and stirred with 1000 rpm for 15 h.The gasket ring between top panel and agitator vessel was removed andswiveled in 32% HCl for 30 seconds, than washed for 30 seconds underfloating VE-H₂O.

Temperature sensor, N₂-pipe, flow brake and agitator shaft are rinsedwith VE-H₂O. The stirrer was removed and disassembled completely. Thedisassembled parts are shown and labeled in FIG. 2. The dissassemblingwas performed by the following steps: i) Remove wire on the top of thestirring engine, ii) pull the agitator shaft out, iii) unscrewed thestirring engine together with the shaft, iv) unscrewed the shaft out ofthe stirring engine (use the steel stick), v) remove gasket ring betweentop panel and shaft, vi) remove circular spring, vii) remove the uppersleeve bearing (use the fork), viii) release the binding screw betweeninner magnet and the agitator shaft and separate the parts, ix) stripdown the lower sleeve bearing, and x) release the agitator blade screwand remove the agitator blade.

The shaft was washed for 30 seconds under floating VE-H₂O. In case ofremaining impurities (brown/dark spots) in the shaft, the shaft wasfilled with HCl and subsequently floated with VE-H₂O. Then, the shaftwas screwed back into the stirring engine. The swing gasket ring betweentop panel and shaft was rinsed for 30 seconds in 32% HCl and floated inVE-H₂O for 30 seconds. The circular spring was washed for 30 secondswith VE-H₂O and bigger particles were removed using forceps.

The inner magnet, binding screw, agitator shaft, stirring blade,stirring blade screw, upper and lower sleeve were rinsed in 30 secondsin 32% HCl and washed after-wards under floating VE-H₂O. The agitatorshaft and the stirring blade are rebuilt with the stirring blade screw.The guide slot was lubricated with glycerin, put on the lower sleevebearing and further lubricated on its outside. The inner magnet and theagitator shaft was assembled with the binding screw. The upper sleevebearing was lubricated in its inside, put it on the inner magnet untilit locks and then lubricated with glycerin on its outside. The flowbreak and the flow break pole was removed, rinsed them together with thescrew nut in 32% HCl for 30 seconds, than wash it under floating VE-H₂Ofor 30 seconds.

The outlet N₂-sleeve was removed. The sleeve clamp was loosened and themetal binding piece was pulled out. The binding piece was rinsed for 30seconds in 32% HCl, then washed under floating VE-H₂O for 30 seconds.Subsequently, the binding piece was inserted in the sleeve and fixedwith the sleeve clamp.

As a next step, manometer, temperature sensor, N₂-pipe, needle valve andburst protection was removed. The respective parts are shown and labeledin FIG. 3. Flush the manometer with VE-H₂O. The burst protection, needlevalve and the N₂-pipe was cleaned with a cleaning cloth soaked in 32%HCl, followed by flushing it with a disposable pipette until the outcoming HCl had no more visible colouring and washing it inside andoutside under floating VE-H₂O for 30 seconds.

The temperature sensor was cleaned with a cleaning cloth soaked in 32%HCl followed by washing it for 30 seconds with VE-H₂O.

The three screws holding the top panel were solved and removed.Subsequently, the three allen keys which fix the core of the top panelwere solved. See FIG. 4 showing the corresponding parts.

The core of the top panel (especially the seven screw holes) was cleanedwith a disposable pipette with 32% HCl. The cleaning step was performeduntil the HCl did not show any visible yellow coloring. The core of thetop panel was then washed under floating VE-H₂O for 30 seconds.

The two pressure sleeves at the agitator vessel were removed. Theagitator vessel was washed for 90 seconds with 32% HCl. Subsequently,the agitator vessel was filled with 100 ml of 32% HCl and cleaned with achemical resistant brush for 60 seconds. The HCl from the agitatorvessel was discarded into a beaker. The last cleaning step was repeatedtwice. The agitator vessel was washed again for 90 seconds with 32% HCl.Finally, the agitator vessel was reassembled with the two pressuresleeves.

The core of the top panel was reassembled with the flow break. Then themanometer, temperature sensor, N2-pipe, needle valve and burstprotection was mounted back into the top panel. The gasket ring wasplaced between the shaft and the top panel. The stirring engine and theagitator shaft was inserted. The gasket ring was placed between toppanel and agitator vessel.

The success of the cleaning procedure was controlled by filling thereassembled reactor with 1 L VE-H₂O. The reactor was heated to 150° C.for 60 minutes with 500 rpm The step was repeated three times. After thethird time of boiling, the VE-H₂O should have been clear, otherwise thecleaning procedure was repeated.

Example 2 Manufacturing of the Ferrimagnetic Iron Oxide Particles

Reaction Equation:3FeCl₂+6NaOH->Fe₃O₄+6NaCl+H₂+2H₂O

400 mL (4.21 mol) of triethylenglycol were put in a glas-reactor. Whilethe reactor was heated to 100° C., stirring with the magnetic stirrerand introducing N₂ (pressure 0.4 bar at the outlet valve) into thetriethylenglycol for 30 minutes was performed. Subsequently, 12.31 gFeCl₂*4H₂O (60.8 mmol) and 12 mL NaOH (10 M) (120 mmol) were added tothe triethylenglycol at 100° C. and still introducing N₂ whilestrengthen stirring (if necessary rise stirring speed) was continued for10 minutes.

Transferring the glas-reactor content into a reactor which was preheatedto 80° C. Stirring was performed for 10 minutes without further heatingand introducing N₂. During continued stirring, the reactor was heatedaccording to the protocol as depicted in FIG. 1. The temperature wasincreased from the preheated temperature of 80° C. to a value of 250° C.within a time period of 20 min. The value of 250° C. was maintained fora time period of 80 min. Subsequently, the second temperature wasdecreased to a value of 30° C. within a time period of 35 min.

After the reactor temperature reached 30° C. the pressure was releasedand the suspension was put into a beaker. The suspension was dialyzedover night in a 36/32 inch sleeve in Milli-Q-H₂O. The sleeve was soakedand washed for 10 minutes with Milli-Q-H₂O before filling it withparticles. After dialyzing over night, the particles were filled into aSchott glas bottle. After sedimentation of the particles and removingthe supernatant to a total volume of 50 mL, the suspension wastransferred into a falkon tube.

Example 3 Silanizing the Ferrimagnetic Iron Oxide Particles

300 mL of a 5.3% w/v silane solution were provided in a special designglas reactor 8 mL of the particle solution (0.5 g particle) asmanufactured according to Example 2 were added. A schematic drawing ofthe special design glas reactor is presented in FIG. 5 (Ar=Argon,M=Motor, US=Ultrasonic probe). Sonification of the solution wasperformed using an ultrasonic probe every 50 seconds 5 times with 80% ofthe maximal amplitude energy intake for 200 ms. Subsequently, 100 mL of1 M HCl was added followed by stirring at 300 rpm and continued use ofthe ultrasonic probe over night (with the same settings as mentionedabove). Then, 10 mL of 1 M HCl was added and the solution was stirredfor two more hours and an argon source drain was attached to the reactorto avoid oxygen in the atmosphere covering the solution. Aftersedimentation, the particles were washed with 400 mL VE-H₂O. After afurther sedimentation of the particles, the supernatant was discarded.Subsequently, 300 mL silane solution was added to the particles.Sonification of the solution was performed every 50 seconds 5 times with80% of the maximal amplitude energy intake for 200 ms. Subsequently, 100mL of 1 M HCl was added followed by stirring at 300 rpm and continueduse of the ultrasonic probe over night (same settings as mentionedabove). Then, 10 mL of 1 M HCl was added and the solution was stirredfor two more hours. After sedimentation, the particles were washed with400 mL VE-H₂O. After a further sedimentation of the particles, thesupernatant was discarded. The washing procedure was repeated 5 moretimes.

Example 4 Use of the Monodisperse Silanized Ferrimagnetic Iron OxideParticles for Independent Generic Nucleic Acid Binding

Monodisperse silanized ferromagnetic iron oxide particles producedaccording to the method described herein as exemplified in the Examples1 to 3 were used for binding target nucleic acids from two differentclinically relevant pathogenic organisms.

The particles exhibited different degrees of silanization, meaning thatdifferent relative amounts of coating were present on the particlesurfaces. More specifically, the different percentages indicated in thetables below represent the mass of coating in relation to the mass ofthe respective iron oxide core. Hence, a suffix of “80%” denotes apopulation of particles wherein the coating mass is 80% of the ironoxide core mass.

Further, one set of particles was coated with two layers of silane,having been subjected to two consecutive rounds of silanization asdescribed herein.

As a reference, magnetic glass particles from the commercially availableMagNA Pure 96 DNA and Viral NA Large Volume Kit (Roche Diagnostics,Catalog No. 06374891001) were used in parallel experiments. Also, thereagents from the above-mentioned kit were used in a nucleic acidisolation procedure following the instructions of the user manual. Theperson skilled in the art is able to readily apply other procedures forusing the particles described herein for binding nucleic acids. It is tobe noted that for each experiment only 1 mg of the particles describedherein were used, while the particles provided with the above-mentionedkit had to be used in portions of 12 mg for each experiment, accordingto the user manual.

After isolating the respective nucleic acids, they were subjected torealtime PCR on a LightCycler® 480 Analyzer (Roche Diagnostics)exploiting the commercially available kits listed in the following.

Isolation of Parvo B19 Virus (DNA)

Equipment:

Name Catalog.No. Company Parvo B 19 Quantification Kit 0324680 809 RocheDiagnosticsPCR results:

Coating Coating Coating Twice Twice to core to core to core Kit Kitsilanized silanized 100% 80% 20% particles particles Particles run1 run2[W/W] [W/W] [W/W] run1 run2 cp-mean 25.37 25.64 26.05 27.86 25.56 26.5425.28 cp-min 25.22 25.59 25.97 27.71 24.99 26.54 25.28 cp-max 25.5225.69 26.10 28.02 25.84 26.54 25.28 Δ cp max − 0.3 0.1 0.1 0.3 0.9 0.00.0 cp min Replicates 2 2 4 3 5 1 1Isolation of Influenza Virus (RNA)Equipment:

Name Catalog No. Company Real Time ready RNA 05 619 416 001 RocheDiagnostics VirusMaster Real Time ready Influenza 05 640 393 001 RocheDiagnostics A H1N1 Detection SetPCR Results:

Coating Coating Coating Twice Twice to core to core to core Kit Kitsilanized silanized 100% 80% 20% particles particles Particles run1 run2[W/W] [W/W] [W/W] run1 run2 cp-mean 29.12 29.14 32.48 34.73 31.31 29.9029.97 cp-min 29.00 29.04 32.29 34.52 31.21 29.84 29.72 cp-max 29.2329.24 32.65 34.90 31.44 29.96 30.22 Δ cp max − 0.2 0.2 0.4 0.4 0.2 0.10.5 cp min Replicates 2 2 4 4 4 2 2

The results clearly show that the particles produced by the methoddescribed herein are at least equivalent to the commercially availablereference particles in their capacity to bind and thereby isolate targetnucleic acids, comprising both DNA and RNA. Notably, the respective Cp(Crossing point) values which, in the current experimental setting,provide a measure for the nucleic acid yield following thebinding/isolation procedure, are overall even improved. This isparticularly evident for the twice-silanized particles produced by themethod described herein, even though in all instances the mass ofparticles used was only a small fraction (1:12) of the mass of prior artparticles.

In summary, it has been shown that the monodisperse silanizedferrimagnetic iron oxide particles of the present invention can be usedfor independent generic nucleic acid binding with properties that are aswell or even better than particles used in the prior art, whiledisplaying further advantageous properties with regard to theirefficiency and their production procedure.

The invention claimed is:
 1. A method for producing a plurality ofsilanized ferromagnetic iron oxide particles for independent genericnucleic acid binding, wherein the method comprises the steps of: a.adding an iron(II) salt to a liquid glycol to obtain a solution, b.raising the pH of the solution to a value of at least 9 such that aprecipitate is obtained, wherein during steps a) and b) a firsttemperature of 20° C. to 120° C. is applied to the solution and whereinduring steps a) and b) the solution is gassed with nitrogen, c. mixingthe solution comprising the precipitate at a second temperature of 150°C. to 300° C. such that ferromagnetic iron oxide particles are obtained,and d. contacting the ferromagnetic iron oxide particles with a silicatesolution such that silanized ferromagnetic iron oxide particles areobtained.
 2. The method of claim 1, wherein contacting the ferromagneticiron oxide particles with the silicate solution comprises the steps of:d1. sonificating the silicate solution comprising the ferromagnetic ironoxide particles, d2. lowering the pH of the silicate solution to a valueof 6 or below such that silanized ferromagnetic iron oxide particles areobtained, d3. washing the silanized ferromagnetic particles with water,and d4. washing the silanized ferromagnetic particles with isopropanolsuch that crosslinking occurs within the silicate layer.
 3. The methodof claim 1, wherein the iron(II) salt is soluble in the liquid glycoland wherein the iron(II) salt is selected from the group consisting ofFeCl₂, FeSO₄, FeAc₂ and the hydrated forms thereof.
 4. The method ofclaim 1, wherein the liquid glycol is triethylene glycol.
 5. The methodof claim 1, wherein the pH of the solution in step b) is raised to avalue of 10.5 using sodium hydroxide.
 6. The method of claim 1, whereinthe first temperature is maintained for 5 to 60 minutes.
 7. The methodof claim 1, wherein the second temperature is maintained for 20 minutesto 48 hours.
 8. The method of claim 1, wherein the silicate is selectedfrom the group consisting of potassium silicate and sodium silicate andwherein the silicate is present in the solution in a concentration from1 to 30% w/v.
 9. The method of claim 2, wherein the pH of the solutionin step d2) is lowered to a value of 5 using hydrochloric acid.
 10. Themethod of claim 1, wherein step d) is repeated one or more times. 11.The method of claim 1, wherein the first temperature is 80° C. to 120°C.
 12. The method of claim 1, wherein the second temperature is 230° C.to 270° C.